Blankenship John W, Varfolomeev Eugene, Goncharov Tatiana, Fedorova Anna V, Kirkpatrick Donald S, Izrael-Tomasevic Anita, Phu Lilian, Arnott David, Aghajan Mariam, Zobel Kerry, Bazan J Fernando, Fairbrother Wayne J, Deshayes Kurt, Vucic Domagoj
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, USA.
Biochem J. 2009 Jan 1;417(1):149-60. doi: 10.1042/BJ20081885.
A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
一类被称为凋亡抑制蛋白（IAP）的抗凋亡调节因子家族可与多种细胞伴侣相互作用，并抑制由多种刺激诱导的细胞凋亡。细胞凋亡抑制蛋白（c-IAP）1和2被招募至肿瘤坏死因子受体1（TNFR1）相关信号复合物，在其中介导受体诱导的核因子κB（NF-κB）激活。此外，c-IAP1和c-IAP2通过其E3泛素连接酶活性，促进NF-κB诱导激酶（NIK）的蛋白酶体降解，并调节非经典NF-κB信号通路。在本文中，我们描述了IAP的一个新型泛素结合结构域。IAP的泛素相关（UBA）结构域分别位于杆状病毒IAP重复（BIR）结构域与c-IAP1和c-IAP2或X连锁凋亡抑制蛋白（XIAP）的半胱天冬酶激活和招募结构域（CARD）或真核生物中一个高度保守的锌指结构域（RING）之间。通过表面等离子体共振或等温滴定量热法测定，c-IAP1的UBA结构域以低微摩尔亲和力结合单泛素以及赖氨酸48（Lys(48)）和赖氨酸63（Lys(63)）连接的多聚泛素链。对c-IAP1的UBA结构域与泛素相互作用的核磁共振（NMR）分析表明，该UBA结构域结合泛素中围绕异亮氨酸44（Ile(44)）的经典疏水补丁。UBA结构域中高度保守的甲硫氨酸-甘氨酸-苯丙氨酸（MGF）结合环内关键氨基酸残基的突变完全消除泛素结合。UBA结构域中的这些突变并未明显影响c-IAP1的泛素连接酶活性，也未影响c-IAP1和c-IAP2参与TNFR1信号复合物。用IAP拮抗剂处理细胞会导致c-IAP1和c-IAP2的蛋白酶体降解。UBA结构域的缺失或突变会减少这种降解，可能是通过减少c-IAP与蛋白酶体的相互作用。这些结果表明，泛素结合可能是自身泛素化的c-IAP1和c-IAP2快速周转的重要机制。
